UBE2C-induced crosstalk between mono- and polyubiquitination of SNAT2 promotes lymphatic metastasis in bladder cancer

Ubiquitination plays an essential role in protein stability, subcellular localization, and interactions. Crosstalk between different types of ubiquitination results in distinct biological outcomes for proteins. However, the role of ubiquitination-related crosstalk in lymph node (LN) metastasis and the key regulatory factors controlling this process have not been determined. Using high-throughput sequencing, we found that ubiquitin-conjugating enzyme E2 C (UBE2C) was overexpressed in bladder cancer (BCa) and was strongly associated with an unfavorable prognosis. Overexpression of UBE2C increased BCa lymphangiogenesis and promoted LN metastasis both in vitro and in vivo. Mechanistically, UBE2C mediated sodium-coupled neutral amino acid transporter 2 (SNAT2) monoubiquitination at lysine 59 to inhibit K63-linked polyubiquitination at lysine 33 of SNAT2. Crosstalk between monoubiquitination and K63-linked polyubiquitination increased SNAT2 membrane protein levels by suppressing epsin 1–mediated (EPN1-mediated) endocytosis. SNAT2 facilitated glutamine uptake and metabolism to promote VEGFC secretion, ultimately leading to lymphangiogenesis and LN metastasis in patients with BCa. Importantly, inhibition of UBE2C significantly attenuated BCa lymphangiogenesis in a patient-derived xenograft model. Our results reveal the mechanism by which UBE2C mediates crosstalk between the monoubiquitination and K63-linked polyubiquitination of SNAT2 to promote BCa metastasis and identify UBE2C as a promising target for treating LN-metastatic BCa.


IHC
For IHC analysis, the paraffin-embedded tissue sections were heated in an incubator for 2 hours at 65°C before being dewaxed with dimethylbenzene and hydrated with gradient alcohols.Subsequently, the sections were placed in heated EDTA buffer to retrieve the antigen and incubated with peroxidase inhibitors to block endogenous peroxidase activity.
After blocking with normal goat serum for 30 min, the sections were incubated with the indicated primary antibodies overnight at 4°C and with secondary antibodies for 30 min at room temperature.Finally, the sections were stained with 3,3'-diaminobenzidine (DAB) and hematoxylin, and images were acquired with a Nikon eclipse 80i (Nikon, Tokyo, Japan) and analyzed with ImageJ software (RRID:SCR_003070).Supplemental Table 7 lists the antibodies used in the experiments.

Histology evaluation of tissue sections
Regarding the examination of UBE2C expression, the percentage of tumor cells that stained positively was classified into the following categories: 0 (no positive staining), 1 (0-10 percent positive), 2 (10-30 percent positive), 3 (30-70 percent positive), and 4 (over 70 percent positive).A four-point rating system was used to indicate the degree of staining: 1 represented no staining, 2 mild staining, 3 moderate staining, and 4 strong staining.A possible score of 0, 1, 2, 3, 4, 6, 8, 9, 12, and 16 could be obtained by multiplying the positive percentage by the staining intensity to calculate the Staining Index (SI).The median value, specifically SI = 8, was then determined to be the threshold.Samples exhibiting low expression were classified as having a SI < 8, while samples exhibiting high expression were classified as having a SI ≥ 8.About the quantification of LYVE1, Image J software (NIH) determined how many vessels had positive staining in three random fields for each section.
The cut off value was used to define the median value.

qRT-PCR
According to the manufacturer's instructions, the total RNA was extracted from the cells by using Total RNA Extraction Reagent (EZBioscience, USA, Cat#EZB-TZ1).Subsequently, the RNA samples were reverse transcribed with the Hiscript III Reverse Transcriptase Kit (Vazyme, Nanjing, China, Cat#R312-01).The expression of the indicated genes was measured by qRT-PCR with the ChamQ TM Universal SYBR qPCR Master Mix Kit (Vazyme, Cat#Q711-02).The detailed sequences of the primers used are listed in Supplemental Table 6.

Transwell assays
Transwell assays were used to evaluate the invasive and migratory ability of BCa cells and the migratory ability of HLECs.To perform migration assays, the lower chamber was filled with 700 μl of medium containing 10% FBS, while the upper chamber (Corning Costar Corp, USA, Cat#3422) was filled with 300 μl of suspension containing either 1 × 10 5 BCa cells or 3 × 10 4 HLECs.To perform invasion assays, the membranes of the upper chambers were first coated with Matrigel (BD Biosciences, USA, Cat#356234).The indicated cells were subsequently plated following the same procedures as those used for the migration assays.
The migrated cells were fixed and stained with 0.1% crystal violet after they had incubated for 4 hours for HLECs, 6 hours for T24 cells, or 12 hours for UM-UC-3 cells at 37°C and 5% CO2.Images were captured with a Nikon eclipse 80i (Nikon, Japan), and the number of migrated cells in five random fields was counted with ImageJ software (RRID:SCR_003070).

Tube formation assays
Tube formation assays were performed to evaluate the tube formation ability of HLECs.
Briefly, 24-well plates were filled with a 400 μl mixture containing Matrigel (BD Biosciences, Cat#356234) and FBS-free ECM at a 1:2 ratio.The plates were then incubated overnight at 37°C.Subsequently, the Matrigel-covered wells were seeded with 300 μl of suspension containing 1 × 10 5 of the indicated HLECs and incubated for 4 hours.Finally, images of the formed lymphatic vessels were obtained via inverted fluorescence microscopy (Olympus IX73, Japan), and the tube length was measured using ImageJ (RRID:SCR_003070).
After the cells were blocked for 1 h at 37°C with normal goat serum, they were incubated with the indicated primary antibody at 4°C overnight.The cells were incubated with the corresponding fluorescent secondary antibodies for 30 min at room temperature, and the nuclei were stained with DAPI for 15 min.Finally, the cells were imaged using laser scanning confocal microscopy (LSM710, Zeiss, Pleasanton, CA, USA).Supplemental Table 7 lists the antibodies used in the experiments.

Flow cytometry
A total of 5 × 10 5 cells were harvested, washed 3 times with PBS and incubated with the Briefly, a 96-well plate was filled with 100 μL of each standard or sample and incubated for 2.5 hours at room temperature.After washing with wash solution, each well, except for the blank wells, was filled with 100 μL of biotinylated VEGFC detection antibody and incubated for 1 hour at 37°C.This was followed by a 45-minute incubation with HRP-streptavidin solution.Finally, the reaction was stopped by adding 50 μl of stop solution, and the OD was measured at 450 nm with a SYNERGY H1 microplate handler (Bio-Tek, USA).

High-throughput sequencing
Total RNA was extracted with TRIzol Reagent (Invitrogen, USA, Cat# 15596026).The mRNA libraries were established and sequenced on a HiSeq 4000 platform by Gene Denovo Biotechnology Co., Ltd.(Guangzhou, China).

PLA
A PLA was used to detect protein interactions in BCa cells.Briefly, BCa cells were seeded in a confocal dish and fixed with 4% paraformaldehyde for 15 minutes.Subsequently, the PLA was performed according to the instructions of the DUOLINK®: PLA Kit (Sigma-Aldrich, Cat#DUO92202) with the indicated antibodies.Images were captured using a laser scanning confocal microscope (Zeiss, USA).

Silver staining
The protein specimens were obtained through coimmunoprecipitation assays, and comparable volumes of these specimens were subjected to electrophoretic separation using a 10% SDS-PAGE gel.Subsequently, the separated proteins underwent a washing process and were subjected to silver staining employing a Silver Stain kit (Thermo Scientific, Cat#24600), expression in high-grade BCa versus low-grade BCa (n = 323).(E) qRT-PCR analysis of UBE2C expression in MIBC versus NMIBC.(F-K) K-M survival analysis of the disease-free survival and overall survival of patients with BCa with low versus high ANLN, SPP1 and TMEM132A expression.The cutoff value is the median.(L) Analysis of the TCGA database revealed UBE2C expression in various human cancers compared with that in normal tissue.(M-O) K-M survival analysis of patients grouped according to UBE2C expression for different cancer types in the TCGA database.(P-R) qRT-PCR analysis of ANLN, SPP1 and TMEM132A expression in LN-positive BCa versus LN-negative BCa (n = 323).The statistical significance of differences was assessed through the nonparametric Mann-Whitney U test in A-E, L and P-R.Data are shown as the mean ± SEM.Supplemental Figure 2. UBE2C promotes the invasion and migration of BCa cells in vitro.(A and B) Western blotting analysis (A) and qRT-PCR analysis (B) of the expression level of UBE2C in 5637, T24, UM-UC-3 and SV-HUC-1 cells.(C and D) qRT-PCR analysis of UBE2C expression following UBE2C overexpression or knockdown in BCa cells.(E-F) Representative images and quantification of the tube formation and migration of HLECs after coculture with UBE2C knockdown or UBE2C-overexpressing T24 cells.Scale bars: 100 μm.(G-J) Representative images and quantification of the migration and invasion of BCa cells after downregulating or overexpressing UBE2C.Scale bars: 100 μm.(K and L) Representative images and quantification of wound healing assays showing the migration capability of UM-UC-3 and T24 cells after downregulation or overexpression of UBE2C.Scale bars: 100 μm.(M and N) Quantification of UBE2C expression in the peritumoral (M) and intratumoral (N) regions of footpad tumor tissues.Significant differences were identified through 1-way ANOVA followed by Dunnett's test in B, C, E, G, I, K and L; 2-tailed Student's t test in D, F, H, J, M and N. Data are shown as the mean ± SEM.Supplemental Figure 4. UBE2C inhibits K63-linked polyubiquitination of SNAT2.(A) Detection of intracellular localization of UBE2C in BCa cells.Scale bars: 5 μm.(B) Immunofluorescence assays showing the colocalization of SNAT2 and UBE2C.Scale bars: 5 μm.(C) Proximity ligation assays showing the interaction between UBE2C and SNAT2.Scale bars: 5 μm.(D) Quantification of the mono-and polyubiquitination levels of SNAT2 after overexpression or knockdown of UBE2C.(E) Quantification of the polyubiquitination levels of SNAT2 after mutation of ubiquitin.(F) IB analysis of the K63-linked polyubiquitination level of SNAT2 after si-UBE2C.Significant differences were identified through 2-tailed Student's t test in D; 1-way ANOVA followed by Dunnett's test in D, E. Data are shown as the mean ± SEM.Supplemental Figure 5. UBE2C blocks the interaction between SNAT2 and NEDD4L.(A) Mass spectrometry analysis of the UBE2C-mediated ubiquitination site in SNAT2.(B) Mass spectrometry analysis of UBE2C-inhibited ubiquitination site in SNAT2.(C) Quantification of ubiquitination level of various lysine residues within SNAT2.(D) IB analysis after co-IP assays with anti-HIS or IgG in UM-UC-3 cells.(E) Proximity ligation assays showing the interaction between SNAT2 and NEDD4L.Scale bars: 5 μm.(F) IB analysis of the monoubiquitination level of SNAT2 after si-UBE2C and si-NEDD4L transfection.(G) IB analysis of the interaction between NEDD4L and SNAT2 after UBE2C overexpression.(H) IB analysis of the interaction between NEDD4L and SNAT2 after 1 SNAT2 K59R mutation.2 Supplemental Figure 6.UBE2C inhibits the endocytosis of SNAT2.(A) IB analysis of SNAT2 expression in BCa cells overexpressing UBE2C.(B) IB analysis of SNAT2 expression in BCa cells after downregulating UBE2C.(C) IB analysis of the half-life of SNAT2 after overexpression of UBE2C.(D and E) FACS analysis with permeabilization and quantification of SNAT2 expression after overexpressing UBE2C.(F-H) Detection of SNAT2 expression in membrane fractions after SNAT2 K59R mutation in UM-UC-3 cells.Scale bar: 5 μm.(I) FACS analysis of the permeabilization and quantification of SNAT2 expression after SNAT2 K59R mutation.(J) FACS analysis with quantification of SNAT2 expression in membrane fractions after overexpressing UBE2C at 20°C.(K) Silver staining for the detection of SNAT2-interacting proteins.(L) Mass spectrometry analysis of SNAT2-binding proteins after co-IP assays.(M) FACS analysis with permeabilization and quantification of SNAT2 expression after overexpressing EPSIN1 and UBE2C.(N) IB analysis of SNAT2 expression in membrane and endosome fractions after overexpression of UBE2C and EPSIN1.Significant differences were identified through 1-way ANOVA followed by Dunnett's test in G, I and M; and 2-tailed Student's t test in E and J. Data are shown as the mean ± SEM.Supplemental Figure 7. UBE2C activates glutamine reprogramming to promote VEGFC secretion.(A and B) Detection of glutamine and glutamate production in the cell extracts for analysis of glutamine uptake by UBE2C-overexpressing T24 cells.(C) Analysis of culture media glutamine in UBE2C-overexpressing T24 cells with a glutamine assay kit.(D) CCK-8 analysis of cell viability in UBE2C-overexpressing T24 cells.(E) Analysis of intracellular ATP production in UBE2C-overexpressing T24 cells with an ATP assay kit.(F-H) Detection of glutamate, cell viability and ATP production in UM-UC-3 cells after SNAT2 K59R mutation.(I) ELISA analysis of VEGFC secretion in T24 cells with overexpression of UBE2C.(J) IB analysis of VEGFC secretion in UM-UC-3 cells with overexpression of UBE2C.(K) ELISA analysis of VEGFC secretion in T24 cells with knockdown of UBE2C.(L) IB analysis of VEGFC secretion in UM-UC-3 cells with knockdown of UBE2C.(M) ELISA analysis of VEGFC secretion after the addition of exogenous glutamine.(N) IB analysis of VEGFC secretion after the addition of exogenous glutamine.(O) ELISA analysis of VEGF-C secretion after the addition of glutamine metabolism inhibitors.(P) IB analysis of VEGF-C secretion after the addition of glutamine metabolism inhibitors.Significant differences were identified through 1-way ANOVA followed by Dunnett's test in C, F, G, H, K, M and O; 2-tailed Student's t test in A, B, D, E and I. Data are shown as the mean ± SEM.Supplemental Figure 8. UBE2C/SNAT2/VEGFC axis is crucial in BCa lymphangiogenesis and LN metastasis.(A-C) Representative images and quantification of tube formation and migration of HLECs treated with culture media from UBE2Coverexpressing T24 cells with or without CB-839 and αVEGFC treatment.Scale bars: 100 μm.(D) qRT-PCR analysis of the UBE2C expression in the tumor tissues from PDXs.(E-H) H-score of UBE2C, SNAT2, VEGFC and LYVE1 positive vessels from PDX tissues.(I-J) Quantification of UBE2C, VEGFC and LYVE1-indicated microlymphatic vessel density in both intratumoral and peritumoral regions of BCa tissues (n = 323).Significant differences were identified through 1-way ANOVA followed by Dunnett's test in B and C; 2-tailed Student's t test in D-H; and the χ 2 test in I and J. Data are shown as the mean ± SEM.